Synthesis and antifungal activity of 2-chloromethyl-1 H -benzimidazole derivatives against phytopathogenic fungi in vitro

Article abstract of DOI:10.1021/jf3053934

A series of 35 benzimidazole derivatives were synthesized from 2-chloromethyl-1H-benzimidazole in good yields. Their structures were characterized by ¹H and ¹³C NMR and HRESIMS. Antifungal activities of all of the synthesized compounds were evaluated against five phytopathogens fungi (Cytospora sp., Colletotrichum gloeosporioides, Botrytis cinerea, Alternaria solani, and Fusarium solani) using the mycelium growth rate method. Compound 4m displayed strong growth inhibition of C. gloeosporioides, A. solani, and F. solani with IC₅₀ of 20.76, 27.58, and 18.60 μg/mL, respectively. Selective inhibition of B. cinerea instead of the other fungal pathogenes was observed with 7f (IC₅₀ of 13.36 μg/mL), comparable to that of positive control, a commercial agricultural fungicide hymexazol (IC₅₀ of 8.92 μg/mL). Compound 5b exhibited remarkable antifungal properties against Cytospora sp., C. gloeosporioides, B. cinerea, and F. solani with IC₅₀ values of 30.97, 11.38, 57.71, and 40.15 μg/mL, respectively; among the target fungi, 5b was the most active compound and superior to the reference against C. gloeosporioides alone. Structure-activity relationship (SAR) data of these compounds are as follows: (1) introduction of the chlorine atom on para-position in the benzene ring help to increase activity (4f vs 4c; 7f vs 7n), (2) the sulfonyl group is critical for the inhibition of C. gloeosporioides (5b and 5c vs 5a), and (3) the unsubstituted benzene ring improve activity (4m vs 4n, 4e and 4a). Thus, compounds 5b, 4m, and 7f emerged as a new leading structure for the development of new fungicides.

Full text of DOI:10.1021/jf3053934

Article
pubs.acs.org/JAFC
Synthesis and Antifungal Activity of 2-Chloromethyl-1H-
benzimidazole Derivatives against Phytopathogenic Fungi in Vitro
Yu-Bin Bai, An-Ling Zhang,* Jiang-Jiang Tang, and Jin-Ming Gao*
Shaanxi Engineering Center of Bioresource Chemistry & Sustainable Utilization, College of Science, Northwest A&F University,
Yangling, Shaanxi 712100, People’s Republic of China
S
* Supporting Information
ABSTRACT: A series of 35 benzimidazole derivatives were synthesized from 2-chloromethyl-1H-benzimidazole in good yields.
1
Their structures were characterized by H and 13C NMR and HRESIMS. Antifungal activities of all of the synthesized
compounds were evaluated against five phytopathogens fungi (Cytospora sp., Colletotrichum gloeosporioides, Botrytis cinerea,
Alternaria solani, and Fusarium solani) using the mycelium growth rate method. Compound 4m displayed strong growth
inhibition of C. gloeosporioides, A. solani, and F. solani with IC50 of 20.76, 27.58, and 18.60 μg/mL, respectively. Selective
inhibition of B. cinerea instead of the other fungal pathogenes was observed with 7f (IC50 of 13.36 μg/mL), comparable to that of
positive control, a commercial agricultural fungicide hymexazol (IC50 of 8.92 μg/mL). Compound 5b exhibited remarkable
antifungal properties against Cytospora sp., C. gloeosporioides, B. cinerea, and F. solani with IC50 values of 30.97, 11.38, 57.71, and
40.15 μg/mL, respectively; among the target fungi, 5b was the most active compound and superior to the reference against
C. gloeosporioides alone. Structure−activity relationship (SAR) data of these compounds are as follows: (1) introduction of the
chlorine atom on para-position in the benzene ring help to increase activity (4f vs 4c; 7f vs 7n), (2) the sulfonyl group is critical
for the inhibition of C. gloeosporioides (5b and 5c vs 5a), and (3) the unsubstituted benzene ring improve activity (4m vs 4n, 4e
and 4a). Thus, compounds 5b, 4m, and 7f emerged as a new leading structure for the development of new fungicides.
KEYWORDS: synthesis, 2-chloromethyl-1H-benzimidazole derivatives, antifungal activity, phytopathogenic fungi,
structure−activity relationship
in use for controlling mycoses in humans.7 Similarly, 2-substituted
INTRODUCTION
■
benzimidazole and its derivatives have been found to be potent
Plant diseases are caused primarily by fungal and bacterial
biologically active compounds as well. The activity and structural
pathogens, leading to severe losses to agriculture and
diversity exhibited by compounds containing the benzimidazole
horticulture crop production worldwide and constitute an
moiety has led to the development of a series of novel and useful
emerging threat to the global food security.1 Considerable
bioactive benzimidazole analogs.9
postharvest losses of fruits and vegetables occur by decay
In our previous work, acetophenone derivatives were designed
caused by fungal pathogens, which, in addition to causing rot,
and synthesized,10 and some compounds showed diverse and
may produce mycotoxins capable of the harmful consequences
promising bioactivities such as fungicidal and antitumor activities.
on animal and human health.2−5 As a result, several important
In continuation of our investigation on the design and synthesis of
fungal plant pathogens of the genera Colletotrichum, Botrytis,
bioactive compounds, a series of novel analogs of 2-chloromethyl-
Alternaria, and Fusarium have received great attention due to
1H-benzimidazole were designed and synthesized from the
their typical infected disease symptoms (e.g., postharvest
intermediates 2-chloromethyl-1H-benzimidazole and 2-chloro-
diseases of fruits such as anthracnose caused by Colletotrichum
methyl-1-methyl-5-nitro-1H-benzimid-azole via an efficient meth-
species).3,4 Many of the currently available antifungal agents
od (Scheme 1). Their antifungal effects on selected five target
have several drawbacks, such as drug related toxicity, severe
phytopathogenic fungi were investigated, and their structure-
drug resistance, nonoptimal pharmacokinetics, and serious drug
antifungal activity relationships were also discussed. To our
interactions.6 Therefore, there is a growing need to develop
knowledge, the antifungal activities of all the synthetic derivatives
new antifungal agents to effectively control those agricultural
including known compounds were reported for the first time.
diseases.
Benzimidazole is one of the commonly used chemical scaffolds
MATERIALS AND METHODS
because it plays an important role in medicinal chemistry and
agrochemicals; it has earned an essential place in the list of
chemotherapeutic agents. During the past 5−10 years, many
condensation products of benzimidazole have been patented for a
variety of biological activities, such as antitumor, antiinflammatory,
antiviral, and antifungal.7−9 Extensive biochemical and pharmaco-
logical studies have confirmed that benzimidazole molecules are
effective against various strains of microorganisms.8 Especially,
benzimidazole is a core substructure of systemic fungicides currently
■
General Experimental Procedures. The melting points of the pro-
ducts were determined on an X-4 apparatus (Beijing Tech Instrument Co.,
Beijing, P. R. China) and are uncorrected. Nuclear magnetic resonance
Received: December 17, 2012
Revised: February 17, 2013
Accepted: February 18, 2013
Published: February 18, 2013
© 2013 American Chemical Society
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a
Scheme 1. Synthetic Route of 2-Chloromethyl-1H-benzimidazole Derivatives
a
Reagents and conditions: (a) HCl, reflux; (b) toluene, (CH3O)2SO2, reflux; (c) triethylamine, acetone, acyl chloride, 0 °C; (d) K2CO3, acetone,
phenols, reflux, 60 °C.
R1=
R2=
4a: −C6H4−COCH3 (m)
4b: −C6H4−COCH3 (o)
4c: −C6H4−COCH3 (p)
4d: −C6H4−COOCH2CH2CH3 (p)
4e: −C6H4−F (p)
5a: −Ac
5b: −O2SCH3
5c: −O2SC6H4Cl(p)
R3=
7a: −C6H4−COCH3 (m)
7b: −C6H4−COCH3 (o)
7c: −C6H4−COCH3 (p)
7d: −C6H4−COOCH2CH2CH3 (p)
4f: −C6H4−Cl (p)
4g: −C6H4−CHO (p)
4h: −C6H3−OCH3 (o)-CH2CHC
H2(p)
4i: −naphthylene (a)
7e: −C6H4−F (p)
7f: −C6H4−Cl (p)
7g: −C6H4−CHO (p)
4j: −C6H4−COOCH2CH3 (p)
4k: −C6H4−COOCH3 (p)
4l: −C6H4−C(CH3)3 (p)
7h: −C6H3−OCH3 (o)-CH2CHC
H2(p)
4m: −C6H5
7i: −naphthylene (a)
4n: −C6H3−(−OCH2O−)(m,p)
4o: −C6H3−OCH3 (o)-CHO(p)
4p: −C6H3−OCH3 (o)-OCCH3(p)
4q: −C6H4−NO2 (p)
7j: −C6H4−COOCH2CH3 (p)
7k: −C6H4−COOCH3 (p)
7l: −C6H4−C(CH3)3 (p)
7m: −C6H5
7n: −C6H3−(−OCH2O−)(m,p)
refluxed in oil bath for 3 h. After cooling, water (10 mL) was added and
the mixture was basified with ammonia.11 After filtration, the solution was
extracted with CHCl3 (3 × 20 mL). The organic layer was dried over
anhydrous Na2SO4 and concentrated. Purification by CC on silica gel
eluting with PE−acetone (4:1) gave the major product 2 as a light yellow
solid (0.55 g, yield 50%) and the minor product 3 as a white crystal
(0.29 g, yield 9%). Compound 2: HR-MS (ESI): m/z calcd. for C9H9ClN2:
181.0527; found: 181.0514 [M+H]+; Compound 3: mp, 126 − 128 °C; 1H
NMR (500 MHz, CDCl3) δ 7.24 (d, J = 7.5 Hz, 1H, ArH), 6.99 (d,
J = 7.8 Hz, 1H, ArH), 6.67 (t, J = 6.7 Hz, 2H, ArH), 4.23 (d, J = 3.2 Hz,
1H, −NH−), 3.84 (q, J = 13.7 Hz, 2H, −CH2Cl−), 3.18 (s, 3H, −NMe−
H), 2.85 (d, J = 4.8 Hz, 3H, −HNMe−H); 13C NMR (125 MHz, CDCl3)
δ 167.7 (−CO), 145.0 (−C−NH−), 130.4 (ArC), 128.0 (ArC), 127.0
(ArC), 117.1 (ArC), 111.3 (ArC), 42.3 (−CH2Cl−), 36.1 (−NMe−C),
30.2 (−HNMe−C); HR-MS (ESI): m/z calcd. for C10H13ClN2O:
213.0789; found: 213.0765 [M+H]+.
(NMR) spectra were recorded on a Bruker Avance spectrometer (Unity
plus 400 or 500 MHz) (Bruker Bios pin, Rheinstetten, Germany) with
tetramethylsilane (TMS) as internal standard. Chemical shift values (δ) were
given in parts per million (ppm). Thin-layer chromatography (TLC) was
performed on silica gel 60 F254 (Qingdao Marine Chemical Ltd., P. R.
China). Column chromatography (CC) was performed over silica gel
(200−300 mesh, Qingdao Marine Chemical Ltd.), High resolution
electrospray ionization mass spectrometry (HRESIMS) data were recorded
on LCMS-IT-TOF (Shimadzu, Kyoto, Japan). Commercial solvents and
reagents were of reagent grade.
General Synthetic Procedure for the Key Intermediates.
Intermediates 1 and 6 were synthesized from o-phenylenediamine
using the reported procedure.11−15 Intermediate 2 was synthesized
according to reported literature.11
Synthesis of 2-Chloromethyl-1H-benzimidazole (1) by Conven-
tional Method. A mixture of o-phenylenediamine (7.56 g, 80 mmol)
and chloroacetic acid (7.56 g, 70 mmol) was taken in a solution of 5 N
HCl (60 mL), refluxed in oil bath for 8 h. The reaction mixture was then
cooled to room temperature and neutralized with ammonia, till neutral to
pH. A yellow residue was obtained by filtration and washed with
water.11,12 Purification by CC on silica gel eluting with petroleum ether
(PE)-acetone (3:1) gave 1 as a yellow solid (9.23 g, yield 79.2%). mp,
152−154 °C (lit.10 154−155 °C); MS (+ESI) m/z 166.96 [M+H]+.
Synthesis of 2-Chloromethyl-1-methyl-benzimidazole (2) by Con-
ventional Method. To a solution of compound 1 (1.01 g, 6.07 mmol)
in anhydrous toluene (10 mL), dimethyl sulfate (0.63 mL, 6.67 mmol)
was added dropwise at room temperature. The reaction mixture was
Synthesis of 2-Chloromethyl-1-methyl-5-nitro-1H-benzimidazole
(6) by Conventional Method. A mixture of 4-nitro-2-amino-1-N-
methylaniline (3.52 g, 21 mmol) and chloroacetic acid (2.58 g, 25.2
mmol) was refluxed for 4 h in 45 mL of 4 N HCl and then cooled to
room temperature and adjusted to pH = 7 with ammonia. A gray
residue was obtained.13−15 Purification by column chromatography on
neutral Al2O3 eluting with CHCl3−MeOH (100:0.8) gave compound
6 as a yellow solid (3.37 g, yield 71%), mp, 180−182 °C (lit.13 191−
1
192 °C). H NMR (500 MHz, CDCl3) δ 8.66 (s, 1H, ArH), 8.28 (d,
J = 8.9 Hz, 1H, ArH), 7.44 (d, J = 8.9 Hz, 1H, ArH), 4.87 (s, 2H,
−CH2O−), 3.96 (s, 3H, Me−H); 13C NMR (125 MHz, CDCl3) δ
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152.9 (ArC), 144.2 (ArC), 141.4 (−N−CN−), 140.2 (ArC), 119.5
(ArC), 117.2 (ArC), 109.8 (ArC), 36.5 (−CH2Cl−), 31.0 (Me-C).
HR-MS (ESI): m/z calcd. for C9H8ClN3O2: 226.0378; found:
226.0352 [M+H]+.
1H NMR (400 MHz, CDCl3) δ 9.83 (s, 1H, ArH), 7.77 (dd, J = 6.9,
1.4 Hz, 1H, ArH), 7.40 (dd, J = 5.6, 1.7 Hz, 2H, ArH × 2), 7.36 (d, J =
8.7 Hz, 2H, ArH × 2), 7.34 − 7.26 (m, 2H, ArH × 2), 5.54 (s, 2H,
−CH2O−), 3.92 (s, 3H, −OMe−H−), 3.91 (s, 3H, −Me−H−). 13C
NMR (100 MHz, CDCl3) δ 191.0 (−CHO), 152.7 (ArC), 150.3
(ArC), 148.6 (ArC), 142.2 (ArC), 136.4 (ArC), 131.2 (ArC), 126.8
(ArC), 123.6 (ArC), 122.6 (ArC), 120.3 (ArC), 113.4 (ArC), 109.6
(ArC), 109.4 (ArC), 64.5 (−CH2O−), 56.1 (−OMe−C), 30.5
(−NMe−C). HR-MS (ESI): m/z calcd. for C17H16N2O3: 297.1234;
found: 297.1220 [M+H]+.
General Procedure To Synthesize Compounds 4a−q by
Conventional Method. To a solution of compound 2 (90.25 mg,
0.5 mmol) and different phenols (0.55 mmol) in dry acetone (30 mL),
anhydrous potassium carbonate (103.5 mg, 0.75 mmol) was added.
The mixture was refluxed in oil bath at 60 °C for 8 h. After cooling and
filtration, the solution was concentrated under vacuum and then
purified by CC on silica gel eluting with CHCl3−MeOH (9:1−95:5)
to obtain the title compounds 4a−q.
General Synthetic Procedure for Compounds 5a−c by a
Slightly Modified Method. To a solution of compound 1 (166.5 mg,
1 mmol) and triethylamine (0.13, 0.9 mmol) in dry acetone (10 mL),
three different acyl chlorides including acetyl chloride, methanesulfonyl
chloride and 4-chlorobenzenesulfonyl chloride (0.9 mmol) was added
dropwise. The mixture was stirred at 0 °C for 12 h.16−18 The mixture
was concentrated under vacuum and then purified by CC on silica gel
eluting with PE−EtOAc (3:1−5:1) to obtain the corresponding
compounds 5a−c.
1-(3-((1-Methyl-1H-benzo[d]imidazol-2-yl)methoxy)phenyl)-
1
ethanone (4a). Yield, 81%; white solid; mp, 128−130 °C; H NMR
(400 MHz, CDCl3) δ 7.82−7.75 (m, 1H, ArH), 7.70−7.64 (m, 1H,
ArH), 7.57 (d, J = 7.6 Hz, 1H, ArH), 7.41−7.34 (m, 2H, ArH × 2),
7.30 (dq, J = 7.0, 5.4 Hz, 3H, ArH × 3), 5.42 (s, 2H, −CH2O−), 3.88
(s, 3H, Me-H), 2.58 (s, 3H, Me-H). 13C NMR (100 MHz, CDCl3) δ
197.9 (−CO), 158.2 (ArC), 149.0 (ArC), 142.3 (ArC), 138.8
(−NC−), 136.3 (ArC), 130.0 (ArC), 123.5 (ArC), 122.6 (ArC),
122.0 (ArC), 120.3 (ArC), 119.9 (ArC), 114.3 (ArC), 109.6 (ArC),
63.5 (−CH2O−), 30.4 (−NMe−C), 26.9 (−Me−C). HR-MS (ESI):
m/z calcd. for C17H16N2O2: 281.1285; found: 281.1264 [M+H]+.
1-(2-((1-Methyl-1H-benzo[d]imidazol-2-yl)methoxy)phenyl)-
2-(Chloromethyl)-1-((4-chlorophenyl)sulfonyl)-1H-benzo[d]-
1
imidazole (5c). Yield, 68%; white solid; mp, 150−152 °C; H NMR
(400 MHz, CDCl3) δ 8.09−8.01 (m, 2H, ArH × 2), 7.92 (t, J = 5.5
Hz, 1H, ArH), 7.78−7.73 (m, 1H, ArH), 7.50 (dd, J = 9.1, 2.1 Hz, 2H,
ArH × 2), 7.45−7.37 (m, 2H, ArH × 2), 5.16 (s, 2H, −CH2−). 13C
NMR (100 MHz, CDCl3) δ 149.0 (C), 142.0 (ArC), 141.7 (ArC),
136.2, 132.9 (ArC), 130.1 (ArC × 2), 129.1 (ArC × 2), 126.5 (ArC),
125.6 (ArC), 121.3 (ArC), 113.6 (ArC), 37.9 (−CH2−). HR-MS (ESI):
m/z calcd. for C14H10Cl2N2O2S: 340.9913; found: 340.9904 [M+H]+.
General Procedure To Synthesize Compounds 7a−n by
Conventional Method. The method to synthesize this series of
compounds is the same as the compound 4.
1
ethanone (4b). Yield, 83%; white solid; mp, 122−124 °C; H NMR
(400 MHz, CDCl3) δ 7.79 (dd, J = 6.9, 1.3 Hz, 1H, ArH), 7.68 (dd, J =
7.7, 1.8 Hz, 1H, ArH), 7.46 (ddd, J = 8.6, 7.4, 1.8 Hz, 1H, ArH), 7.40−
7.36 (m, 1H, ArH), 7.36−7.28 (m, 3H, ArH × 3), 7.08−6.99 (m, 1H,
ArH), 5.48 (s, 2H, −CH2O−), 3.90 (s, 3H, Me−H), 2.55 (s, 3H, Me−H).
13C NMR (100 MHz, CDCl3) δ 199.6 (−CO), 156.9 (ArC), 148.8
(ArC), 142.1 (−NC−), 136.3 (ArC), 133.8 (ArC), 130.5 (ArC), 129.0
(ArC), 123.7 (ArC), 122.7 (ArC), 121.8 (ArC), 120.3 (ArC), 113.3 (ArC),
109.7 (ArC), 63.8 (−CH2O−), 31.8 (−NMe−C), 30.5 (Me−C). HR-MS
(ESI): m/z calcd. for C17H16N2O2: 281.1285; found: 281.1266 [M+H]+.
1-(4-((1-Methyl-1H-benzo[d]imidazol-2-yl)methoxy)phenyl)-
1-(3-((1-Methyl-5-nitro-1H-benzo[d]imidazol-2-yl)methoxy)-
phenyl)ethanone (7a). Yield, 82%; white solid; mp, 200−202 °C; 1H
NMR (400 MHz, CDCl3) δ 8.62 (d, J = 2.0 Hz, 1H, ArH), 8.19 (dd,
J = 8.9, 2.1 Hz, 1H, ArH), 7.60 (s, 1H, ArH), 7.53 (d, J = 7.7 Hz, 1H,
ArH), 7.41 − 7.31 (m, 2H, ArH × 2), 7.22 (dd, J = 8.2, 2.6 Hz, 1H,
ArH), 5.39 (s, 2H, −CH2O−), 3.90 (s, 3H, Me-H), 2.53(s, 3H, Me).
13C NMR (100 MHz, CDCl3) δ 197.7 (−CO), 158.0 (ArC), 153.0
1
ethanone (4c). Yield, 86%; white solid; mp, 186−188 °C; H NMR
(400 MHz, CDCl3) δ 8.00−7.86 (m, 2H), 7.78 (dd, J = 6.8, 1.5 Hz,
1H, ArH), 7.38−7.27 (m, 3H, ArH × 3), 7.19−7.08 (m, 2H, ArH × 2),
5.44 (s, 2H, −CH2O−), 3.87 (s, 3H, Me−H), 2.53 (s, 3H, Me−H); 13
C
(ArC), 144.0 (ArC), 141.6 (−NC−), 140.3 (ArC), 138.9 (ArC),
130.2 (ArC), 122.5 (ArC), 119.9 (ArC), 119.3 (ArC), 117.1 (ArC),
114.0 (ArC), 109.7 (ArC), 63.4 (−CH2O−), 31.0 (−NMe−C), 26.9
(−OC−Me−C). HR-MS (ESI): m/z calcd. for C17H15N3O4:
326.1135; found: 326.1126 [M+H]+.
NMR (100 MHz, CDCl3) δ 196.8 (−CO), 161.7 (ArC), 148.8 (ArC),
142.3 (−NC−), 136.3 (ArC), 131.3 (ArC), 130.8 (ArC × 2), 123.6
(ArC), 122.6 (Ph- C), 120.3 (ArC), 114.7 (ArC × 2), 109.6 (ArC), 63.5
(−CH2O−), 30.4 (−NMe−C), 26.5 (Me−C). HR-MS (ESI): m/z calcd.
for C17H16N2O2: 281.1285; found: 281.1265 [M+H]+.
1-(2-((1-Methyl-5-nitro-1H-benzo[d]imidazol-2-yl)methoxy)-
phenyl)ethanone (7b). Yield, 81%; white solid; mp, 202−204 °C; 1H
NMR (400 MHz, Pyr) δ 8.89 (d, J = 2.1 Hz, 1H, ArH), 8.30 (dd, J =
9.0, 2.2 Hz, 1H, ArH), 7.88 (dd, J = 7.7, 1.7 Hz, 1H, ArH), 7.52 (dd,
J = 8.3, 3.4 Hz, 2H, ArH × 2), 7.50 − 7.44 (m, 1H, 1H, ArH), 7.09 −
7.02 (m, 1H, 1H, ArH), 5.70 (s, 2H, −CH2O−), 3.88 (s, 3H, Me−H),
2.62 (s, 3H, Me−H). 13C NMR (100 MHz, C5D5N) δ 198.8 (−C
O), 157.2 (ArC), 153.6 (ArC), 143.9 (ArC), 141.9 (−NC−), 140.7
(ArC), 133.7 (ArC), 130.5 (ArC), 129.3 (ArC), 121.8 (ArC), 118.8
(ArC), 116.8 (ArC), 113.8 (ArC), 110.6 (ArC), 63.5 (−CH2O−), 31.6
(−NMe−C), 30.6 (−OC−Me−C). HR-MS (ESI): m/z calcd. for
C17H15N3O5: 326.1135; found: 326.1128 [M+H]+.
Propyl 4-((1-methyl-1H-benzo[d]imidazol-2-yl)methoxy)-
1
benzoate (4d). Yield, 80%; white solid; mp, 96−98 °C; H NMR
(400 MHz, CDCl3) δ 8.10−7.92 (m, 2H, ArH × 2), 7.82−7.75 (m,
1H, ArH), 7.42−7.24 (m, 3H, ArH × 3), 7.20−7.04 (m, 2H, ArH ×
2), 5.44 (s, 2H, −CH2O−), 4.23 (t, J = 6.7 Hz, 2H, −CH2O−), 3.88
(s, 3H, Me-H), 1.85−1.68 (m, 2H, −CH2−), 1.00 (t, J = 7.4 Hz, 3H,
Me−H). 13C NMR (100 MHz, CDCl3) δ 166.3 (ArC), 161.5 (ArC),
148.8 (ArC), 142.2 (ArC), 136.3 (ArC), 131.8 (ArC × 2), 124.2
(ArC), 123.6 (ArC), 122.6 (ArC), 120.3 (ArC), 114.6 (ArC × 2),
109.6 (ArC), 66.5 (−CH2O−), 63.5 (−CH2O−), 30.5 (−NMe−C),
22.3 (−CH2−), 10.6 (Me-C). HR-MS (ESI): m/z calcd. for
C9H9ClN2: 325.1547; found: 325.1526 [M+H]+.
1-(4-((1-Methyl-5-nitro-1H-benzo[d]imidazol-2-yl)methoxy)-
1
phenyl)ethanone (7c). Yield, 85%; white solid; mp, 254−256 °C; H
2-((Benzo[d][1,3]dioxol-5-yloxy)methyl)-1-methyl-1H-benzo[d]-
1
imidazole (4n). Yield, 80%; light pink solid; mp, 138−140 °C; H
NMR (400 MHz, C5D5N) δ 8.92 (d, J = 2.2 Hz, 1H, ArH), 8.31 (dd,
J = 8.9, 2.2 Hz, 1H, ArH), 8.09 (d, J = 8.9 Hz, 2H, ArH × 2), 7.50 (d,
J = 9.0 Hz, 1H, ArH), 7.34 (d, J = 8.9 Hz, 2H, ArH × 2), 5.69 (s, 2H,
−CH2O−), 3.81 (s, 3H, Me−H), 2.51 (s, 3H, Me-H). HR-MS (ESI):
m/z calcd. for C17H15N3O6: 326.1135; found: 326.1128 [M+H]+.
Propyl-4-((1-methyl-5-nitro-1H-benzo[d]imidazol-2-yl)methoxy)-
NMR (400 MHz, CDCl3) δ 7.77 (dd, J = 6.9, 1.5 Hz, 1H, ArH), 7.32
(dtd, J = 9.2, 7.1, 1.5 Hz, 3H, ArH × 3), 6.69 (d, J = 8.5 Hz, 1H), 6.62
(d, J = 2.5 Hz, 1H, ArH), 6.51 (dd, J = 8.5, 2.5 Hz, 1H, ArH), 5.90 (s,
2H, −OCH2O−), 5.30 (d, J = 1.8 Hz, 2H, −CH2O−), 3.87 (d, J = 2.1
Hz, 3H, −Me−H−). 13C NMR (100 MHz, CDCl3) δ 152.4 (ArC),
148.5 (ArC), 147.5 (ArC), 141.6 (ArC), 141.3 (−C−), 135.3 (ArC),
122.4 (ArC), 121.5 (ArC), 119.2 (ArC), 108.5 (ArC), 107.2 (ArC),
105.3 (ArC), 100.4 (ArC), 97.6 (−OCH2O−), 63.5 (−CH2O−), 29.4
(−NMe−C). HR-MS (ESI): m/z calcd. for C16H14N2O3: 283.1077;
found: 283.1059 [M+H]+.
1
benzoate (7d). Yield, 83%; white solid; mp, 140−142 °C; H NMR
(400 MHz, CDCl3) δ 8.68 (d, J = 2.1 Hz, 1H, ArH), 8.25 (dd, J = 9.0,
2.1 Hz, 1H,ArH), 8.05−7.98 (m, 2H, ArH × 2), 7.43 (d, J = 9.0 Hz,
1H, ArH), 7.14−7.06 (m, 2H, ArH × 2), 5.47 (s, 2H, −CH2O−), 4.24
(t, J = 6.7 Hz, 2H, −OCH2Et), 3.96 (s, 3H, Me−H), 1.76 (dq, J =
14.2, 7.2 Hz, 2H, −CH2Me), 1.00 (t, J = 7.4 Hz, 3H, Me−H). 13C
NMR (100 MHz, CDCl3) δ 166.2 (−CO), 161.2 (ArC), 152.7
3-Methoxy-4-((1-methyl-1H-benzo[d]imidazol-2-yl)methoxy)-
benzaldehyde (4o). Yield, 88%; light yellow solid; mp, 144 − 146 °C;
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(ArC), 144.1 (ArC), 141.6 (−NC−), 140.3 (ArC), 131.9 (ArC × 2),
124.6 (ArC), 119.3 (ArC), 117.1 (ArC), 114.5 (ArC × 2), 109.7 (ArC),
66.6 (−CH2O−), 63.3 (−CH2Et), 31.1 (−NMe−C), 22.2 (−CH2Me),
10.6 (Me-C). HR-MS (ESI): m/z calcd. for C19H19N3O5: 370.1397;
found: 370.1384 [M+H]+.
2.2 Hz, 1H, ArH), 8.24−8.12 (m, 2H, ArH × 2), 7.49 (d, J = 9.0 Hz,
1H, ArH), 7.42−7.30 (m, 2H, ArH × 2), 5.67 (s, 2H, −CH2O−), 3.80
(s, 6H, Me × 2-H). HR-MS (ESI): m/z calcd. for C17H15N3O5:
342.1084; found: 342.1079 [M+H]+.
2-((4-(tert-Butyl)phenoxy)methyl)-1-methyl-5-nitro-1H−benzo-
[d]imidazole (7l). Yield, 85%; yellow solid; mp, 158−160 °C; 1H
NMR (400 MHz, CDCl3) δ 8.69 (d, J = 2.0 Hz, 1H, ArH), 8.25 (dd,
J = 8.9, 2.1 Hz, 1H, ArH), 7.42 (d, J = 9.0 Hz, 1H, ArH), 7.39−7.27 (m,
2H, ArH × 2), 7.06−6.97 (m, 2H, ArH × 2), 5.40 (s, 2H, −CH2O−),
3.99 − 3.93 (m, 3H, Me−H), 1.29 (s, 9H, Me × 3-H). 13C NMR (100
MHz, CDCl3) δ 155.4 (ArC), 153.7 (ArC), 145.0 (ArC), 144.0 (ArC),
141.5 (−NC−), 140.4 (ArC), 126.7 (ArC 2), 119.2 (ArC), 117.0
(ArC), 114.3 (ArC × 2), 109.6 (ArC), 71.0 (−CH2O−), 34.3 (−C−
Me3), 31.6 (Me × 3-C), 31.1 (−NMe−C). HR-MS (ESI): m/z calcd. for
C17H15N3O5: 340.1656; found: 340.1642 [M+H]+.
2-((4-Fluorophenoxy)methyl)-1-methyl-5-nitro-1H-benzo[d]-
1
imidazole (7e). Yield, 80%; light yellow solid; mp, 224−226 °C; H
NMR (400 MHz, CDCl3) δ 8.69 (d, J = 2.1 Hz, 1H,ArH), 8.27 (dd,
J = 8.9, 2.1 Hz, 1H, ArH), 7.43 (d, J = 9.0 Hz, 1H, ArH), 7.07−6.93
(m, 4H, ArH × 4), 5.38 (s, 2H, −CH2O−), 3.96 (s, 3H, Me−H). 13
C
NMR (100 MHz, CDCl3) δ 159.3 (ArC), 153.8 (ArC), 153.2 (ArC),
144.1 (ArC), 141.60 (−NC−), 140.4 (ArC), 119.3 (ArC), 117.1
(ArC), 116.5 (ArC), 116.3 (ArC), 116.1 (ArC), 116.1 (ArC), 109.6
(ArC), 64.0 (−CH2O−), 31.1 (−NMe−C). HR-MS (ESI): m/z calcd.
for C15H12FN3O3: 302.0935; found: 302.0925 [M+H]+.
1-Methyl-5-nitro-2-(phenoxymethyl)-1H-benzo[d]imidazole
(7m). Yield, 82%; light yellow solid; mp, 234−236 °C; 1H NMR (400
MHz, CDCl3) δ 8.70 (d, J = 2.1 Hz, 1H, ArH), 8.27 (dd, J = 9.0, 2.1
Hz, 1H, ArH), 7.43 (d, J = 9.0 Hz, 1H, ArH), 7.32 (dd, J = 8.5, 7.5 Hz,
2H, ArH × 2), 7.07 (d, J = 8.0 Hz, 2H, ArH × 2), 7.02 (t, J = 7.4 Hz,
1H, ArH), 5.42 (s, 2H, −CH2O−), 3.97 (s, 3H, Me−H). 13C NMR
(100 MHz, CDCl3) δ 157.6 (ArC), 153.3 (ArC), 143.9 (ArC), 141.5
(−NC−), 140.3 (ArC), 129.8 (ArC × 2), 122.1 (ArC), 119.1
(ArC), 116.9 (ArC), 114.7 (ArC × 2), 109.5 (ArC), 63.2 (−CH2O−),
31.0 (−NMe−C). HR-MS (ESI): m/z calcd. for C15H13N3O3:
284.1030; found: 284.1016 [M+H]+.
2-((4-Chlorophenoxy)methyl)-1-methyl-5-nitro-1H-benzo[d]-
1
imidazole (7f). Yield, 81%; white solid; mp, 256−258 °C; H NMR
(400 MHz, C5D5N) δ 8.90 (d, J = 2.1 Hz, 1H, ArH), 8.30 (dd, J = 8.9,
2.2 Hz, 1H, ArH), 7.48 (d, J = 9.0 Hz, 1H, ArH), 7.41−7.32 (m, 2H,
ArH × 2), 7.24−7.21 (m, 2H, ArH × 2), 5.56 (s, 2H, −CH2O−), 3.78
(s, 3H, Me−H). HR-MS (ESI): m/z calcd. for C15H12ClN3O3:
318.0640; found: 318.0633 [M+H]+.
4-((1-Methyl-5-nitro-1H-benzo[d]imidazol-2-yl)methoxy)-
1
benzaldehyde (7g). Yield, 83%; white solid; mp, 248−250 °C; H
NMR (400 MHz, C5D5N) δ 10.02 (s, 1H, −CHO), 8.92 (d, J = 2.1
Hz, 1H, ArH), 8.31 (dd, J = 9.0, 2.2 Hz, 1H, ArH), 7.96 (d, J = 8.8 Hz,
2H, ArH × 2), 7.50 (d, J = 9.0 Hz, 1H, ArH), 7.38 (d, J = 8.7 Hz, 2H,
ArH × 2), 5.70 (s, 2H, −CH2O−), 3.81 (s, 3H, Me-H). HR-MS (ESI):
m/z calcd. for C16H13N3O4: 312.0979; found: 312.0968 [M+H]+.
2-((4-Allyl-2-methoxyphenoxy)methyl)-1-methyl-5-nitro-1H-
benzo[d] imidazole (7h). Yield, 83%; light yellow solid; mp, 114−116
2-((Benzo[d][1,3]dioxol-5-yloxy)methyl)-1-methyl-5-nitro-1H-
benzo[d]imidazole (7n). Yield, 81%; light yellow solid; mp, 246−248
1
°C; H NMR (400 MHz, C5D5N) δ 8.88 (d, J = 2.1 Hz, 1H, ArH),
8.29 (dd, J = 8.9, 2.2 Hz, 1H, ArH), 7.47 (d, J = 8.9 Hz, 1H, ArH),
6.98 (d, J = 2.5 Hz, 1H, ArH), 6.86 (d, J = 8.5 Hz, 1H, ArH), 6.74 (dd,
J = 8.5, 2.5 Hz, 1H, ArH), 5.94 (s, 2H, −OCH2O−), 5.52 (s, 2H,
CH2O), 3.80 (s, 3H, Me-H). HR-MS (ESI): m/z calcd. for
C16H13N3O5: 328.0928; found: 328.0921 [M+H]+.
1
°C; H NMR (400 MHz, CDCl3) δ 8.66 (d, J = 2.1 Hz, 1H, ArH),
8.24 (dd, J = 8.9, 2.1 Hz, 1H, ArH), 7.41 (d, J = 9.0 Hz, 1H, ArH),
7.03 (d, J = 8.1 Hz, 1H, ArH), 6.77−6.67 (m, 2H, ArH × 2), 5.93 (ddt,
J = 16.9, 10.1, 6.7 Hz, 1H, −CH2CH−), 5.41 (s, 2H, −CH2O−), 5.15
− 5.01 (m, 2H, −CH2), 4.02 (s, 3H, MeO-H), 3.84 (s, 3H, Me−H),
3.32 (d, J = 6.7 Hz, 2H, −CH2CH−). 13C NMR (100 MHz, CDCl3) δ
153.8 (ArC), 150.1 (ArC), 145.3 (ArC), 143.9 (ArC), 141.6 (−N
C−), 140.5 (ArC), 137.4 (ArC), 135.5 (ArC), 120.8 (ArC), 119.1
(ArC), 117.00 (ArC), 116.1 (CH2), 115.9 (ArC), 112.6 (ArC),
109.5 (ArC), 65.2 (−CH2O−), 55.9 (MeO-C), 40.0 (−CH2−), 31.0
(−NMe−C). HR-MS (ESI): m/z calcd. for C19H19N3O4: 354.1448;
found: 354.1436 [M+H]+.
For spectral data of known compounds 1, 2, 4e−m, 4p−q, 5a−b,
and 6 see the Supporting Information.
Antifungal Bioassay. According to our previously reported
methods,10,19−21 fungicidal activities of the synthetic compounds
were tested in vitro against five plant pathogenic fungi (Cytospora sp.,
Colletotrichum gloeosporioides, Botrytis cinerea, Alternaria solani, and
Fusarium solani) by using the mycelium growth rate method. All of the
fungi were provided by the Institute of Pesticides, Northwest A&F
University. The strains were retrieved from the storage tube and
cultured for 2 weeks at 28 °C on potato dextrose agar (PDA).
Antifungal activity was assessed as follows: the synthesized compounds
were screened in vitro for their antifungal activities against the five
phytopathogenic fungi. PDA medium was prepared in the flasks and
sterilized. Those compounds were dissolved in acetone before mixing with
molten agar at 55 °C, and final concentrations of the stock solutions of
the tested compounds dissolved in acetone were 100, 50, 25, 12.5, and
6.25 μg/mL. The medium was then poured into sterilized Petri dishes. All
types of fungi were incubated in PDA at 28 °C for 1 week to get new
mycelium for the antifungal assays, and a mycelia disk of approximately
4 mm diameter cut from culture medium was picked up with a sterilized
inoculation needle and inoculated in the center of the PDA Petri
dishes.5,14 The inoculated Petri dishes were incubated at 28 °C for 3−4 d.
Acetone was served as the control, while commercially available agricultural
fungicide hymexazol was used as a positive control. Each sample was
measured by three replicate, each colony diameter of the three replicate was
measured 4 times by cross bracketing method. After the mycelia grew
completely, the diameters of the mycelia were measured and the inhibition
rate was calculated according to the formula
1-Methyl-2-((naphthalen-1-yloxy)methyl)-5-nitro-1H-benzo[d]-
1
imidazole (7i). Yield, 82%; pink solid; mp, 200−202 °C; H NMR
(400 MHz, CDCl3) δ 8.78 − 8.67 (m, 1H, ArH), 8.34 − 8.17 (m, 2H,
ArH × 2), 7.82 (d, J = 7.7 Hz, 1H, ArH), 7.63−7.46 (m, 3H, ArH ×
3), 7.40 (dd, J = 18.9, 8.6 Hz, 2H, ArH × 2), 7.11 (d, J = 7.6 Hz, 1H,
ArH), 5.62 (s, 2H, −CH2O−), 4.00 (s, 3H, Me−H). 13C NMR (100
MHz, CDCl3) δ 153.4 (ArC), 153.3 (ArC), 144.0 (ArC), 141.7
(−NC−), 140.5 (ArC), 134.7 (ArC), 127.9 (ArC), 126.8 (ArC),
125.9 (ArC), 125.9 (ArC), 125.4 (ArC), 121.9 (ArC), 121.5 (ArC),
119.2 (ArC), 117.1 (ArC), 109.6 (ArC), 105.6 (ArC), 63.5
(−CH2O−), 31.2 (−NMe−C). HR-MS (ESI): m/z calcd. for
C19H15N3O3: 334.1186; found: 334.1177 [M+H]+.
Ethyl 4-((1-methyl-5-nitro-1H-benzo[d]imidazol-2-yl)methoxy)-
1
benzoate (7j). Yield, 83%; white solid; mp, 190−192 °C; H NMR
(400 MHz, CDCl3) δ 8.68 (d, J = 2.0 Hz, 1H, ArH), 8.26 (dd, J = 8.9,
2.1 Hz, 1H, ArH), 8.01 (d, J = 8.8 Hz, 2H, ArH × 2), 7.43 (d, J = 9.0
Hz, 1H, ArH), 7.10 (d, J = 8.9 Hz, 2H, ArH × 2), 5.47 (s, 2H,
−CH2O−), 4.34 (q, J = 7.1 Hz, 2H, −OCH2CH3−), 3.96 (s, 3H, Me−
H), 1.36 (t, J = 7.1 Hz, 3H,Me−H). 13C NMR (100 MHz, CDCl3) δ
166.2 (ArC), 161.2 (ArC), 152.7 (ArC), 144.1 (ArC), 141.56 (−N
C−), 140.3 (ArC), 131.9 (ArC × 2), 124.6 (ArC), 119.3 (ArC), 117.1
(ArC), 114.5 (ArC × 2), 109.7 (ArC), 63.3 (−CH2O−), 61.0
(−OCH2Me), 31.1 (−NMe−C), 14.48 (Me−C). HR-MS (ESI): m/z
calcd. for C18H17N3O5: 356.1241; found: 356.1231 [M+H]+.
inhibition rate (%) = (C − T)/(C − 4mm) × 100%
where C is the average diameter of mycelia in the blank test, and T is the
average diameter of mycelia on treated PDA with those compounds. The
inhibition ratio of those compounds at the concentration of 100 μg/mL is
summarized in Table 1. The IC50 (median inhibitory concentration)
values of some compounds were determined, and the results were listed
in Table 2.
Methyl 4-((1-methyl-5-nitro-1H-benzo[d]imidazol-2-yl)methoxy)-
1
benzoate (7k). Yield, 81%; white solid; mp, 246−248 °C; H NMR
(400 MHz, C5D5N) δ 8.94−8.89 (m, 1H, ArH), 8.30 (dd, J = 8.9,
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by a second nucelophilic substitution to form 3. Subsequent
Williamson reaction of the resulting intermediate 2 with various
substituted phenols in dry acetone in anhydrous K2CO3 produced
the desired compounds 4a−q in good yields (>80%). Furthermore,
treatment of 1 in the presence of triethylamine with three acetyl
chloride and two sulfonyl chlorides yielded the corresponding
products 5a−c in ca. 70% yield. Similarly, the target compounds
7a−n were prepared from 6 in good yields (>80%). At the
beginning of this study, we made attempts to prepare 6 from 2
through nitration reaction, but an inseparable mixture of two
isomers, 6 and its 6-nitro-1H-benzimidazole, was obtained by
column chromatography.
The structures of the synthetic compounds were confirmed
by 1H NMR, 13C NMR and MS, as well as HRESIMS
spectroscopic data. Out of the 35 synthesized derivatives, 22
compounds (3, 4a−d, 4n, 4o, 5c, and 7a−n) were new. It
should be noted that the synthetic approach for this family of
compounds used in the current study is a simple, efficient and
low-cost method with high yields.
Antifungal Activity. Preliminary in vitro screening results
of the title compounds for antifungal activities against five
phytopathogenic fungi, Cytospora sp., Colletotrichum gloeospor-
ioides, Botrytis cinerea, Alternaria solani, and Fusarium solani, are
listed in Table 1. The results indicated that some of these
synthetic compounds exhibited over 65% growth inhibition
against mycelial growth of these tested fungi at concentrations
of 100 μg/mL. Here, the inhibition rates were for only IC50
values for further comparison.
Encouraged by these preliminary findings, we planned
further SAR studies on the title compounds. We determined
their IC50 (median inhibitory concentration) values by using
mycelial growth inhibitory rate method. As shown in Table 2,
the tested compounds presented different fungicidal activity
against Cytospora sp. and C. gloeosporioides, superior to the
positive control hymexazol (IC50 > 100 μg/mL), whereas they
were inactive or less active than hymexazol against B. cinerea
and A. solani (IC50 = 8.92 and 14.03 μg/mL, respectively).
Specifically, a fungicidal activity of the intermediate 6, bearing a
−NO2 group, was 2−3 fold of 1 and 2 against the five fungal
species (except B. cinerea). Notably, 6 showed remarkable
antifungal activities against Cytospora sp., C. gloeosporioides, A.
solani, and F. solani (IC50 = 22.48, 28.03, 27.95, and 23.34 μg/mL,
respectively). The results revealed that the presence of the NO2
group at the 5-position of benzimidazole ring in 6 could be critical
for antifungal activity.
Table 1. Preliminary Antifungal Activities of Compounds at
100 μg/mL
a
average values of inhibition rate (%) to five pathogens
compound
C. s.
C. g.
B. c.
A. s.
F. s.
6.59
1
2
<5.00
45.40
<5.00
55.46
64.52
87.07
48.28
80.60
76.29
20.98
49.71
62.64
79.89
31.03
28.16
84.20
80.60
10.20
26.01
19.54
8.05
67.57
69.57
41.27
41.27
71.43
38.10
20.63
62.67
90.48
49.21
38.10
22.22
71.74
<5.00
63.84
87.30
80.95
<5.00
14.29
41.27
43.87
100.00
74.60
100.00
<5.00
<5.00
<5.00
<5.00
<5.00
6.35
53.22
91.53
64.39
82.46
64.91
65.41
<5.00
47.37
83.05
<5.00
47.37
<5.00
35.90
6.43
25.29
51.56
25.29
64.52
65.52
55.67
63.62
85.22
77.83
27.75
40.07
50.74
39.08
64.62
23.65
75.37
63.98
40.89
17.90
11.33
11.33
67.98
31.86
100.00
<5.00
13.79
33.50
8.05
28.76
30.11
78.49
64.73
81.85
42.20
83.87
64.07
11.96
60.35
52.96
21.37
48.25
<5.00
84.54
74.46
24.73
<5.00
8.60
3
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
64.81
29.82
88.30
15.20
59.06
<5.00
57.26
79.86
57.26
48.70
<5.00
<5.00
38.60
<5.00
<5.00
100.00
<5.00
23.98
<5.00
<5.00
32.20
57.26
7.89
4m
4n
4o
4p
4q
5a
5b
5c
6
21.37
79.17
28.09
100.00
<5.00
<5.00
<5.00
19.35
9.95
100.00
27.44
100.00
51.87
<5.00
<5.00
10.92
22.41
10.20
9.48
7a
7b
7c
7d
7e
7f
<5.00
30.21
16.26
35.14
19.54
11.33
<5.00
30.21
10.51
7.22
<5.00
<5.00
25.40
<5.00
<5.00
18.01
<5.00
5.91
7g
7h
7i
<5.00
<5.00
<5.00
<5.00
59.13
30.16
<5.00
<5.00
49.21
<5.00
<5.00
<5.00
<5.00
<5.00
<5.00
12.36
38.94
7j
7k
7l
7m
7n
Hy
91.23
100.00
34.81
72.45
80.30
a
A.s., Alternaria solani; B.c., Botrytis cinerea; C.g., Colletotrichum
gloeosporioides; C.s. Cytospora sp; F.s., Fusarium solani; Hy, hymexazol.
In case of the 4-series, compound 4m, with nonsubstituents
in the benzene ring displaying increased effects, showed higher
growth inhibition of the three fungi C. gloeosporioides, A. solani,
and F. solani with IC50 values of 20.76, 27.58, and 18.60 μg/mL
(Table 2), respectively, than other 4-series derivatives such as
4e and 4l with a fluorine or t-butyl group, but had no activity
toward B. cinerea. This indicated that para- or ortho-
substitution of the benzene ring is not beneficial for the
activity. Notably, 4m showed a fungicidal activity against F.
solani which was 2-fold of the control hymexazol (IC50 of 38.49
μg/mL). We reasoned that F. solani was not sensitive to the Cl
atom (4f vs 4m and 4e). Furthermore, within the 4-series
derivatives, compound 4f, having a chlorine atom on para-
position of the benzene ring, was the best compound in
inhibiting both Cytospora sp. and B. cinerea with IC50 values of
24.91 and 26.82 μg/mL, respectively, in particular, with
inhibition of 4f on Cytospora sp. being much higher than that
of the control hymexazol. This suggested the presence of this
The data of the fungicidal activities were statistically analyzed using
Excel software from Windows to give the results of IC50 values. The
results were expressed as the mean SD of triplicate experiments
RESULTS AND DISCUSSION
■
Chemistry. The synthetic routes of the title compounds are
outlined in Scheme 1. Reaction of o-phenylenediamine with
chloroacetic acid under reflux gave compound 1 in 79% yield,
which was then treated with dimethyl sulfate in dried toluene to
introduce a methyl group at N-1 position, affording the correspond-
ing benzimidazole 2 as a major product in 50% yield and 2-chloro-
N-(2-(methylamino)phenyl)acetamide 3 as a minor one. A possible
formation mechanism of 3 was shown in Scheme 2. A formation of
benzimidazole was thought to be reversible,22 so a primary amine
group of the resulting amide intermediate proceeds a first
nucelophilic substitution reaction with dimethyl sulfate followed
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a
Table 2. Antifungal Activity of Some Compounds against Five Phytopathogens
IC50 SD/ (μg/mL)
compound
C. s.
C. g.
B. c.
A. s.
F. s.
1
2
NA
>100
83.15 12.63
80.29 2.03
>120
>100
68.11 3.70
68.88 5.90
>100
>100
>100
NA
>150
4a
4b
4c
4e
4f
>100
>100
82.36 5.54
>100
>100
70.62 0.81
>120
>100
50.68 1.17
58.25 1.34
24.91 0.34
52.58 1.53
50.04 1.03
54.30 1.44
NA
89.58 9.54
>120
>100
39.05 7.08
31.52 1.41
>100
>100
69.35 4.51
62.46 1.10
>150
41.26 2.25
49.71 3.58
20.76 0.67
55.69 0.18
>150
26.82 2.77
>150
4j
>150
4m
4n
5a
5b
5c
6
>150
27.58 0.59
>100
18.60 1.10
72.97 0.84
NA
63.72 10.03
50.91 2.65
57.71 3.51
>100
NA
30.97 1.12
NA
11.38 0.90
22.29 0.69
28.03 1.69
NA
>100
40.15 0.76
NA
NA
22.48 0.44
NA
>100
27.95 3.52
NA
23.34 2.08
NA
7f
13.36 1.35
69.10 2.57
8.92 0.44
7n
Hy
NA
NA
NA
NA
>100
>100
14.03 0.14
38.49 6.95
a
NA, not active.
Both 7f and 7n presented much higher inhibitory activity against
B. cinerea than the parent compound 6. This suggested that the
chlorine atom at para-position in the phenyl ring of 7f plays an
important role in the fungicidal activity to B. cinerea. Intriguingly,
in comparison with 6, 7f, and 7n had no fungicidal activity against
the other pathogenic species tested Cytospora sp., C. gloeospor-
ioides, A. solani, and F. solani since 7f and 7n were devoid of the
chlorine group of 6. These findings indicated that B. cinerea is
more susceptible to 7f than to 7n and that the chlorine atom of 6
to be a structural feature important for improved antifungal activity
to the other organisms.
In conclusion, we have reported the synthesis of a series of
2-chloromethyl-1H-benzimidazole derivatives as well as the
ability of these compounds to inhibit growth of five fungal phyto-
pathogenes tested. Many of the synthetic compounds tested,
including intermediate 6, were found to have significant antifungal
activity against certain phytopathogenic fungi in vitro. Compounds
4m and 6 displayed higher antifungal activity against Cytospora
sp., C. gloeosporioides, A. solani, and F. solani. 5c and 7f also
exhibited strong antifungal properties with relatively high selectivity
against C. gloeosporioides and B. cinerea with IC50 values of 22.29
and 13.36 μg/mL, respectively. Compound 5b gave substantially
strong antifungal effects on C. gloeosporioides with an IC50 value of
11.38 μg/mL, which is much better than that of hymexazol, a well-
known commercial fungicide. Taken together, compounds 4m, 5b,
and 7f were demonstrated to possess the most potent inhibition of
the fungal plant pathogens tested in the present study and could be
potential lead structures for further discovery of novel antifungal
agrochemicals.
Scheme 2. Possible Mechanism for the Formation of 3
chlorine atom on para-position in the benzene ring to
contribute to enhancing activity. Additionally, compared to
intermediate 2, displacement of the chlorine atom of 2 by
varying phenoxyl groups resulted in the general trend of
improving inhibitory activity of 4-series compounds to the five
fungi due to lower IC50 data (Table 2). For example, inhibitory
potency of 4m to C. gloeosporioides increased by approximately
4-fold of 2 with an IC50 of 80.29 μg/mL.
In case of the 5-series, 5b with a methanesulfonyl group
addition exhibited moderate to strong antifungal effects on
Cytospora sp., C. gloeosporioides, B. cinerea, and F. solani with IC50
values of 30.97, 11.38, 57.71, and 40.15 μg/mL, respectively, while
5c selectively inhibited growth of C. gloeosporioides with an IC50
value of 22.29 μg/mL but was almost inactive toward the other
fungal pathogens. Notably, 5b was 2-fold more active against C.
gloeosporioides (IC50 = 11.38 μg/mL) than 5c with a 4-chloro-
benzenesulfonyl moiety, which were both superior to the reference
(IC50 > 100 μg/mL). In addition, 5a gave moderate activity
toward B. cinerea with an IC50 value of 50.91 2.65 μg/mL but
had no antifungal effects on other fungal pathogens. These
findings indicated that introduction of a small bulky sulfonyl group
at the NH-position of the benzimidazole ring appeared to be more
favorable for increasing activity against C. gloeosporioides (5b and
5c vs 5a). These structures provide new templates for the
potential treatment and management of anthracnose diseases.
However, only two phenoxyl ether derivatives 7f and 7n of
intermediate 6 selectively inhibited the mycelial growth of B.
cinerea (IC50 of 13.36 and 69.10 μg/mL, respectively), with 7f
being 5-fold stronger than 7n and being comparable to
hymexazol (IC50 = 8.92 μg/mL) in antifungal potency (Table 2).
ASSOCIATED CONTENT
* Supporting Information
HRMS and NMR spectra of new synthetic compounds, and
(HR)MS and NMR data of known compounds. This material is
available free of charge via the Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
*Tel: +86-29-87092335; e-mail: anlingzh@nwsuaf.edu.cn;
jinminggao@nwsuaf.edu.cn.
■
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dx.doi.org/10.1021/jf3053934 | J. Agric. Food Chem. 2013, 61, 2789−2795

Journal of Agricultural and Food Chemistry
Article
molecular hydrogen-bond formation in aqueous solution between (N-
1)H and the phosphonate group. Org. Biomol. Chem. 2003, 1, 1819−
1826.
(18) Okamoto, Y.; Ueda, T. Acylation of 2-cyanomethylbenzimida-
zole. Chem. Pharm. Bull. 1977, 11, 3087−3089.
(19) Li, H.-Q.; Li, X.-J.; Wang, Y.-L.; Zhang, Q.; Zhang, A.-L.; Gao,
J.-M.; Zhang, X.-C. Antifungal metabolites from Chaetomium globosum,
an endophytic fungus in Ginkgo biloba. Biochem. Syst. Ecol. 2011, 39,
876−879.
Funding
This work was financially supported by grants from the
National Natural Science Foundation of China (No.
30971882), the Program of Natural Science Basis Research in
Shaanxi (No. 2009JM3010), and Shaanxi Province Science and
Technology (No. 2011k02−07).
Notes
The authors declare no competing financial interest.
(20) Li, X. J.; Zhang, Q.; Zhang, A. L.; Gao, J. M. Metabolites from
Aspergillus fumigatus, an endophytic fungus associated with Melia
azedarach, and their antifungal, antifeedant, and toxic activities. J. Agric.
Food Chem. 2012, 60, 3424−3431.
(21) Li, X. J.; Shi, X. W.; Shuai, Q.; Gao, J. M.; Zhang, A. L. Bioactive
metabolites from biotransformation of paeonol by the white-rot
basidiomycete Coriolus versicolor. Nat. Prod. Commun. 2011, 6, 1129−
1130.
REFERENCES
■
(1) Savary, S.; Teng, P. S.; Willocquet, L.; Nutter, , F. W., Jr
Quantification and modeling of crop losses: A review of purposes.
Annu. Rev. Phytopathol. 2006, 44, 89−112.
(2) Brase, S.; Encinas, A.; Keck, J.; Nising, C. F. Chemistry and
̈
biology of mycotoxins and related fungal metabolites. Chem. Rev. 2009,
109, 3903−3990.
(22) Wright, J. B. The chemistry of the benzimidazoles. Chem. Rev.
1951, 48, 397−541.
(3) Meepagala, K. M.; Kuhajek, J. M.; Sturtz, G. D.; Wedge, D. E.;
Vulgarone, B. The antifungal constituent in the steam-distilled fraction
of Artemisia douglasiana. J. Chem. Ecol. 2003, 29, 1771−1780.
(4) Pan, Z. Z.; Zhu, Y. J.; Yu, X. J.; Lin, Q. F.; Xiao, R. F.; Tang, J. Y.;
Chen, Q. X.; Liu, B. Synthesis of 4′-thiosemicarbazonegriseofulvin and
its effects on the control of enzymatic browning and postharvest
disease of fruits. J. Agric. Food Chem. 2012, 60, 10784−10788.
(5) Saremi, H.; Okhovvat, S. M. Mycotoxin producing Fusarium
species associated with plant disease on potato, wheat, corn and animal
diseases in northwest Iran. Commun. Agric. Appl. Biol. Sci. 2006, 71,
1175−1185.
(6) Xu, H.; Fan, L.-L. Antifungal agents. Part 4: Synthesis and
antifungal activities of novel indole [1,2-c]-1,2,4-benzotriazine
derivatives against phytopathogenic fungi in vitro. Eur. J. Med. Chem.
2011, 46, 364−369.
(7) Amrutkar, S. V., Sunil V; Bhagat, U. D; Pargharmol., P.; Kotgire,
S. S. Synthesis and antifungal activity of 1alkyl/h2[4(alkyl/
arylpiperazin1yl)methyl]benzimidazole derivatives. Int. J. Pharm.
Pharm. Sci. 2010, 2, 84−92.
(8) Ansari, K. F.; Lal, C. Synthesis, physicochemical properties and
antimicrobial activity of some new benzimidazole derivatives. Eur. J.
Med. Chem. 2009, 44, 4028−4033.
(9) Vaidya, S. D.; Kumara, B. V. S.; Kumar, U. N. B. R. V. Synthesis,
anti-bacterial, anti-asthmatic and anti-diabetic activities of novel N-
substituted-2-(benzo[d]isoxazol-3-ylmethyl)-1H-benzimidazoles. J.
Heterocyclic Chem. 2007, 44, 685−691.
(10) Ma, Y. T.; Fan, H. F.; Gao, Y.; Li, H.; Zhang, A. L.; Gao, J. M.
Natural products as sources of new fungicides (I): Synthesis and
antifungal activity of acetophenone derivatives against phytopatho-
genic fungi. Chem. Biol. Drug. Des. 2012, DOI: 10.1111/cbdd.12064.
(11) Njoya, Y.; Boufatah, N.; Gellis, A.; Rathelot, P. Microwave-
assisted synthesis of new benzimidazoles. Heterocycles 2002, 57, 1423−
1432.
(12) Sheng, C.; Che, X.; Wang, W.; Wang, S. Design and synthesis of
antifungal benzohetercyclic derivatives by scaffold hopping. Eur. J.
Med. Chem. 2011, 46, 1706−1712.
(13) Skolnik, H.; Miller, J. G.; Day, A. R. The allylic character of 2-(a-
chloroalky1)-benzimidazoles. J. Am. Chem. Soc. 1943, 65, 1854−1858.
(14) Popov, I. I.; Narezhnaya, V. N.; Zubenko, A. A. Research on
unsaturated azole derivatives. Khim. Geterotsikl. Soedin. 1978, 8, 1104−
1107.
(15) Khan, K. M.; Shah, Z.; Ahmad, V. U.; Ambreen, N.; Khan, M.;
Taha, M.; Rahim, F.; Noreen, S.; Perveen, S.; Choudhary, M. I.;
Voelter, W. 6-Nitrobenzimidazole derivatives: Potential phosphodies-
terase inhibitors: Synthesis and structure-activity relationship. Bioorg.
Med. Chem. 2012, 20, 1521−1526.
(16) RamLa, M. M.; Omar, M. A.; El-Khamry, A. M.; El-Diwani, H. I.
Synthesis and antitumor activity of 1-substituted-2-methyl-5-nitro-
benzimidazoles. Bioorg. Med. Chem. 2006, 14, 7324−7332.
(17) Sanchez-Moreno, M. J.; Gomez-Coca, R. B.; Fernandez-Botello,
A.; Ochocki, J. Synthesis and acid-base properties of (1H-
benzimidazol-2-yl-methyl)phosphonate (Bimp2-). Evidence for intra-
2795
dx.doi.org/10.1021/jf3053934 | J. Agric. Food Chem. 2013, 61, 2789−2795